Abstract The rat glucose-dependent insulinotropic peptide (GIP) gene has been isolated and characterized. The gene spans approximately 8.2 kilobase pairs (kb) and the GIP mRNA (0.8 kb) is encoded by six exons. The 42 amino acid hormone is encoded by exons 3 and 4. The exon-intron organization of the rat GIP gene revealed that the splice acceptor site for intron 2 is 24 nucleotides downstream compared to the comparable splice acceptor site in the human gene. This intron sliding results in an 8 amino acid deletion in the amino terminal extension of the prepropeptide. Primer extention analysis and RNase protection assay demonstrated the existence of multiple closely spaced sites for transcriptional initiation. Both the 5′-flanking region and intron 1 contain TATA and CCAAT boxes consistent with initiation of gene transcription, although a TATA box in intron 1 is functionally inactive in adult rats in spite of its reasonable location.